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Background: Interpretation of serial high-sensitivity cardiac troponin (hs-cTn) measurements for the diagnosis of acute myocardial infarction (AMI) assumes random fluctuation of hs-cTn around an individual's homeostatic set point. The aim of this study was to challenge this diagnostic concept., Methods: Study 1 examined the presence of a diurnal hs-cTn rhythm by hourly blood sampling, day and night, in 24 individuals without a recent history of AMI. Study 2 assessed morning vs evening diagnostic accuracy of hs-cTnT and hs-cTnI in a prospective multicenter diagnostic study of 2782 unselected patients, presenting to the emergency department with acute chest pain., Results: In study 1, hs-cTnT, but not hs-cTnI, exhibited a diurnal rhythm, characterized by gradually decreasing concentrations throughout daytime, rising concentrations during nighttime, to peak concentrations in the morning (mean 16.2 ng/L at 8:30 AM and 12.1 ng/L at 7:30 PM). In study 2, the hs-cTnT rhythm was confirmed by higher hs-cTnT concentrations in early-morning presenters compared to evening presenters with an adjudicated diagnosis of noncardiac disease. The diagnostic accuracy [area under the receiver-operation characteristics curve (AUC)] of hs-cTnT at presentation, 1 h, and for the combination of absolute changes with presenting concentration, were very high and comparable among patients presenting early morning as compared to evening (all AUC >0.93). hs-cTnI exhibited no diurnal rhythm with no differences in AUC among early-morning and evening presenters., Conclusions: Rhythmic diurnal variation of hs-cTnT is a general phenomenon that is not seen with hs-cTnI. While the diurnal hs-cTnT rhythm does not seem to affect the diagnostic accuracy of hs-cTnT for AMI, it should be considered when using hs-cTnT for screening purposes., Clinical Trial Registration: 1. Circadian Variation of Cardiac Troponin, NCT02091427, www.clinicaltrials.gov/ct2/show/NCT02091427. 2. Advantageous Predictors of Acute Coronary Syndrome Evaluation (APACE) Study, NCT00470587, www.clinicaltrials.gov/ct2/show/NCT00470587., (© 2016 American Association for Clinical Chemistry.)